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Journal of Virology, June 2000, p. 5516-5524, Vol. 74, No. 12
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Reovirus Nonstructural Protein µNS Binds to Core Particles but Does Not Inhibit Their Transcription and Capping Activities

Teresa J. Broering,1,2 Aimee M. McCutcheon,1,2 Victoria E. Centonze,3,dagger and Max L. Nibert1,2,*

Department of Biochemistry,1 Institute for Molecular Virology,2 and Integrated Microscopy Resource,3 University of Wisconsin-Madison, Madison, Wisconsin 53706

Received 23 August 1999/Accepted 22 March 2000

Previous studies provided evidence that nonstructural protein µNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455-466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of µNS. Given the possible role of µNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant µNS can bind to cores in vitro. The µNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of µNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of µNS were bound. Thus, the µNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the µNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to µNS. The µNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing µNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins µ1 and sigma 3. These findings are consistent with the presence of µNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, µNS may block or delay outer-capsid assembly and allow continued transcription by these particles.

* Corresponding author. Mailing address: Institute for Molecular Virology, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 262-4536. Fax: (608) 262-7414. E-mail: mlnibert{at}facstaff.wisc.edu.

dagger Present address: UTHSCSA, San Antonio, TX 78284.

Journal of Virology, June 2000, p. 5516-5524, Vol. 74, No. 12
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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